1. Field of the Invention
This invention is in the field of mass spectrometric analysis of multiple immune system modulators in a complex mixture. Immune system modulators typically have molecular masses of less than about 30 kilo Daltons, and include chemokines and cytokines (which encompass interferons and interleukins) and arachidonic acid metabolites.
2. Description of the Related Art
There is a great need in the biotechnology industry for a method that would permit the rapid and accurate identification of immune system modulators in a complex mixture, for example, a biological sample. Such immune system modulators include chemokines and cytokines (which encompass using interferons and interleukins). Immune modulators also include arachidonic acid metabolites and truncated receptors for immune modulators secreted by infected cells or pathogens to manipulate the host immune system, such as virokines, and other pathogen-encoded proteins that affect the host immune system, host cell response molecules, and peptide fragments.
Cytokines are a varied group of proteins that are released by mammalian cells and act on other cells through specific receptors through which they elicit a wide variety of responses affecting the immune system. Cytokine actions include control of cell proliferation and differentiation, regulation of immune responses, hemopoiesis and inflammatory responses. Included among the cytokines are growth factors, interferons, interleukins and tumor necrosis factors. The majority of cytokines has molecular masses below 30 kilo Daltons in their monomeric form, and most have molecular masses in the ranges of 8,000–10,000 Daltons or 15,000–20,000 Daltons. A Dalton is a unit of atomic and molecular mass, equal to one-twelfth of the mass of the nuclide carbon-12. It is identical with the unified atomic mass unit.
Chemokines are other immune system modulators that constitute a super family of soluble proteins implicated in a wide range of acute and inflammatory processes and other immunoregulatory functions.
Chemokines and cytokines are nearly indistinguishable on SDS PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gels based on the relative molecular mobility at this level of resolution. The most widely used method for identifying cytokines in a complex mixture is the Enzyme Linked Immunosorbent Assay (ELISA), which antigenically distinguishes cytokines (Balkwill, Cytokines—a practical approach. Oxford University Press, New York. 1991). ELISAs are expensive and time consuming, taking more than 90 minutes to complete. Moreover, there is significant cross-sensitivity between a given antibody to a cytokine and other closely related cytokines in the same class which causes inaccuracies, for example among the interferons and interleukins. Further, ELISAs only permit analysis of one cytokine at a time, therefore a separate ELISA has to be run to detect each unique cytokine in a complex mixture.
Even mass spectrometry with its high resolving power for low molecular mass species, cannot identify cytokines and other immune system modulators in a complex mixture.
Many studies have been reported showing that infection of an animal by a pathogen causes dramatic changes in the expression of one or more immune modulators in the animal at the site of infection. However, the techniques available for detecting the presence of immune system modulators in a complex mixture are slow, expensive, sometimes unreliable, and technically difficult to perform, thus making it impractical to monitor changes in immune modulators in biological samples in real time as a diagnostic tool for diagnosing infectious diseases in large numbers of people such as might be infected in a terrorist attack using biological weapons. A rapid and accurate method for detecting the presence or absence of immune modulators in biological samples in real time or near real time would open the door for developing new tools to diagnose infections, and for monitoring disease progression.
Based on the foregoing, there is a clear need for a rapid, sensitive and affordable method for detecting multiple immune system modulators in a complex mixture.